Updated publication reference for PubMed record(s): 31556620. For the YPIC challenge 2018 contestants were invited to try to decipher two unknown English questions encoded by a synthetic protein expressed in E. coli. We present how we analyzed this unknown sample using a tryptic digest with dynamic exclusion disabled to increase the signal-to-noise ratio of the measured molecules. Subsequently, spectral clustering was used to generate high-quality consensus spectra and condense the acquired MS/MS spectral data. De novo spectrum identification was used to determine the English questions encoded by the synthetic protein, and any post-translational modifications introduced by E. coli on the synthetic protein were detected using spectral networking. Although the synthetic protein sample for the YPIC challenge 2018 is not of biological interest, the experimental and computational strategy presented here can be directly used to analyze samples for which no protein sequence information is available. All software and code to perform the bioinformatics analysis is available as open source, and a self-contained Jupyter notebook is provided to fully recreate the analysis.